The objective of this project is to understand the structure-function relationship of phospholipase A2 (PLA2) at the highest possible resolution. in the past eight years we have investigated the structural and functional properties of >100 site-specific mutants and made several major advances. The new thrust is to expand on three themes and seven specific aims. Theme I is to extend and complete previous studies on the structural and functional roles of the hydrogen bonding network (Specific Aim 1) and the disulfide bonds (Specific Aim 2), by deleting multiple hydrogen bonds and multiple disulfide bonds, respectively. Theme II (Specific Aim 3) is to map the interfacial binding site by fluorescence quenching. It has been established that binding of PLA2 to the interface makes Trp-3 inaccessible to fluorescence quenching by water-soluble acrylamide or succinimide. The single tryptophan residue, Trp-3, will be moved around the surface of PLA2 by making 20-30 double mutants W3F/XnW. The mutants will be used to map out the residues involved in binding to an anionic interface and a zwitterionic interface. Theme III consists of three specific aims centering on the structure-function relationship of PLA2 from the structural perspective. Preliminary studies suggested that many PLA2 mutants are converted to a semi-molten globule state" (sMG state) in a pH- dependent way. Specific Aim 4 is to characterize the sMG state of these mutants by NMR, CD, and fluorescence. Specific Aim 5 is to examine the structural changes (upon addition of acid) that may lead to the onset of the conversion to the sMG state, and to understand why WT and some mutants differ so much in the transition pH. Five specifdic properties will be examined: 3 degree structure, backbone dynamics, conformational stability, pKa of side chain carboxylates, and calcium binding affinity. Specific Aim 6 is to design mutants with different degrees of structural perturbations and different transitions pH, and characterize their structures and functions. Specific Aim 7 is to continue and enhance the collaboration with M. Jain on interfacial kinetics and with M. Sundaralingam on x-ray crustallography.